ABSTRACT Painful diabetic neuropathy (PDN) is one of the most common and intractable symptoms of diabetes, affecting 25% of diabetic patients1-4. The hallmarks of PDN are neuropathic pain and small fiber degeneration, manifested by the loss of dorsal root ganglion (DRG) nociceptor axons5,6. Neuropathic pain is associated with nociceptor hyper-excitability in the absence of physiologically appropriate stimuli7-9. However, the molecular mechanisms leading to the hyper-excitability of DRG nociceptors and neuropathic pain in PDN are unknown, as are the mechanisms leading to small fiber degeneration. This fundamental gap in our knowledge represents a critical barrier to progress in developing novel therapeutic approaches for PDN. Indeed current drugs available for treating pain have not proved to be particularly effective for treating PDN3,24 and the long term effects of drugs like opioids, in particular, are highly problematic3. It would therefore be of great therapeutic significance if novel drugs could be developed for treating PDN associated pain that are based on a clear understanding of the molecular factors that are responsible for producing the symptoms associated with the disease. Moreover, given the fact that G-protein coupled receptors (GPCRs) have been shown to be particularly ?druggable? targets, GPCRs (such as the CXCR4 receptor) that are specifically associated with the development of the symptoms of PDN would represent particularly interesting targets for the development of new, effective, non-opioid, and not additive therapeutics for PDN. A major aim of our grant proposal entitled ?Cellular and Molecular Role of CXCR4 signaling in Painful Diabetic Neuropathy? was to use an unbiased approach for identifying genes whose expression was specifically associated with the natural history of PDN. Since the start of the grant funding period we have initiated these studies and have already made some extremely interesting observations suggesting novel therapeutic targets associated with the development of the disease. In particular transcriptomal analysis of dorsal root ganglion (DRG) neurons taken from high fat diet (HFD) fed mice which express the major symptoms of PDN10, demonstrate changes in a set of genes which all cluster in association with a specific DRG neuronal subtype expressing the Mas-related G protein-coupled receptor D (MrgprD) that has previously been implicated in neuropathic pain11,12. We have therefore, designated this set of genes the ?MrgprD Associated Cluster? (MAC). The objective of this application is to rigorously validate the functional role of MrgprD signaling in the pathogenesis of nociceptor excitability, neuropathic pain and small fiber degeneration in PDN. MrgprD is an excitatory receptor that has significant constitutive activity13, suggesting that small molecules that have inverse agonist activity at this receptor might be of particular use in the treatment of PDN. Our central hypothesis is that MrgprD signaling is driving nociceptor hyper-excitability leading to neuropathic pain and small fiber degeneration in PDN. We will test this central hypothesis through the following specific aims: AIM1. Determine the regulation of MrgprD and associated MAC genes during the natural history of PDN in mice. Our initial results, which identified MAC genes associated with the MrgprD expressing DRG population, compared control mice with those in which PDN was completely established after 10 weeks of HFD treatment. We wish to map the detailed pattern of the expression of these genes over the complete time course of the disease, starting right after the initiation of HFD treatment. These data are important as they will tell us precisely when MrgprD and other MAC genes might be targeted for maximal effect. Moreover, specific targeting of transcriptomal analysis to the isolated MrgprD expressing population would be predicted to allow more detailed analysis of changes in gene transcription associated with the development of PDN. AIM2. Determine of the effects of manipulating MrgprD signaling on the symptoms of PDN. In order to make a compelling case for MrgprD receptors as a target for the treatment of PDN associated pain, it is vital to demonstrate that manipulation of these receptors produces a reduction in the symptoms of PDN. We will examine this possibility using several experimental approaches. First, we will test the effects of activating inhibitory DREADD receptors targeted to the MrgprD expressing population of DRG neurons. Secondly, we will examine the effects of reducing the expression of MrgprD receptors using conditional knockout mouse genetics and CRISPR based approaches. The CRISPR based approach can also be used to examine the consequences manipulating any of the MAC associated genes. Endpoints to be examined will include the development of pain hypersensitivity behavior and the dying back of cutaneous sensory innervation typically associated with PDN. AIM3. Validate MrgprD as potential target for the treatment of PDN. Because MrgprD is a GPCR that exhibits a significant degree of constitutive activity, it is predicted that its overexpression, even in the absence of an activating ligand, would produce neuronal hyperexcitability that would lead to pain hypersensitivity behavior. If this is the case then an MrgprD inverse agonist should prove to be therapeutically useful in the treatment of PDN associated pain. We will examine this possibility by testing small molecules with these characteristics on the excitability of DRG neurons and the symptoms of PDN following their administration in vivo. The identification of MrgprD receptors as a promising novel target for the treatment PDN associated pain is the result of the confluence of our previously published approach to this problem together with our current unbiased transcriptomal approach and therefore constitutes and extremely intriguing lead.